There are more than 40 million people infected by the HIV virus worldwide where approximately 5 million new infections occurred during 2005. Worldwide, approximately 1 in every 100 adults aged 15 to 49 is infected with HIV. Although antiviral therapy can extend the life of individuals, the death toll continues to rise: 3 million people died from AIDS last year. Although antiviral nucleoside analog therapy successfully delays progression of HIV infection to AIDS, these drugs cause unwelcome side effects by inducing mitochondrial toxicity. Current antiviral nucleoside analog therapy against HIV results in compromised mitochondrial function due to selective inhibition of the mitochondrial DNA polymerase. As much as 20% of patients undergoing AZT treatment develop a mitochondrial dysfunctional disease known as red ragged fiber disease and D4T and ddC cause neuropathy in 15-20% of patients. The mode and effect of antiviral nucleotide analogs, by AZT, ddI, 3TC, D4T and others on the inhibition and fidelity of the mitochondrial DNA polymerase and mitochondrial DNA replication are poorly understood. What structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to its inhibition patterns is poorly understood. We previously compared the inhibition, insertion, and exonucleolytic removal of five currently approved antiviral nucleotide analogs on the purified human recombinant DNA polymerase gamma. The apparent Km and kcat values were determined for the incorporation of TTP, dCTP, dGTP, 2'-3'-dideoxy-TTP (ddTTP), 3'-azido-TTP (AZT-TP), 2'-3'-dideoxy-CTP (ddCTP), 2!?-3!?didehydro-TTP (D4T-TP), (-)-2',3'-dideoxy-3'-thiacytidine (3TC-TP), and carbocyclic 2',3'-didehydro-dGTP (CBV-TP). Human pol gamma readily incorporated all five analogs into DNA but with varying efficiencies. Kinetic studies indicate that the apparent in vitro hierarchy of mitochondrial toxicity for the approved NRTIs is: ddC(zalcitabine) !Y ddI(didanosine) !Y D4T(stavudine) > >3TC(lamivudine) >PMPA(tenofovir)> AZT(zidovudine) > CBV(abacavir). The human pol gamma utilized dideoxynucleotides and D4T-TP in vitro as efficiently as the natural deoxynucleoside triphosphates, whereas AZT-TP, 3TC-TP and CBV-TP were moderate inhibitors of chain elongation. With the exception of terminally incorporated 3TC, the pol gamma 3!?-5!? exonuclease was inefficient at removing these five analogs from DNA and removal required enzyme levels exceeding substrate concentrations. Even though discrimination against inserting AZT and CBV makes them only moderate inhibitors in vitro, their inefficient excision suggest AZT and CBV may persist in vivo once incorporated into mtDNA by pol gamma. Finally, we found that the exonuclease activity is inhibited by AZT-monophosphate at concentrations known to occur in cells. Thus, although these analogs exert their greatest effect by insertion and chain termination of DNA synthesis, the persistence in DNA and inhibition of proofreading activity may also contribute to mitochondrial toxicity.[unreadable] The mitochondrial deoxynucleotide carrier (DNC) transports nucleotide precursors (or phosphorylated NRTIs) into mitochondria for mitochondrial (mt)DNA replication or inhibition of mtDNA replication by NRTIs. Transgenic mice (TG) expressing human DNC targeted to murine myocardium we used to define mitochondrial events from NRTIs in vivo and findings were corroborated by biochemical events in vitro. Zidovudine (3'-azido-2',3'-deoxythymidine; ZDV), stavudine (2', 3'-didehydro-2', 3'-deoxythymidine; d4T), or lamivudine ((-)-2'-deoxy-3'-thiacytidine; 3TC) were administered individually to TGs and wild-type (WT) littermates (35 days) at human doses with drug-free vehicle as control. Left ventricle (LV) mass was defined echocardiographically, mitochondrial ultrastructural defects were identified by electron microscopy, the abundance of cardiac mtDNA was quantified by real time polymerase chain reaction, and mtDNA-encoded polypeptides were quantified. Untreated TGs exhibited normal LV mass with minor mitochondrial damage. NRTI monotherapy (either d4T or ZDV) increased LV mass in TGs and caused significant mitochondrial destruction. Cardiac mtDNA was depleted in ZDV and d4T-treated TG hearts and mtDNA-encoded polypeptides decreased. Changes were absent in 3TC-treated cohorts. In supportive structural observations from molecular modeling, ZDV demonstrated close contacts with K947 and Y951 in the DNA pol gamma active site that were absent in the HIV reverse transcriptase active site. We concluded that NRTIs deplete mtDNA and polypeptides, cause mitochondrial structural and functional defects in vivo, follow inhibition kinetics with DNA pol gamma in vitro, and are corroborated by molecular models. Disrupted pools of nucleotide precursors and inhibition of DNA pol gamma by specific NRTIs are mechanistically important in mitochondrial toxicity.